Effect of temperature and growth media on the attachment of Listeria monocytogenes to stainless steel

Listeria monocytogenes ATCC 19111 cultivated in nutrient-rich medium (brain heart infusion, BHI) or starved in minimal medium (10% filter sterilized pond water and 90% sterilized distilled water) were investigated for their initial attachment to austenitic stainless steel No. 4 with satin finish at 4 °C, 20 °C, 30 °C, 37 °C, or 42 °C. A droplet (10 μl) containing  10⁷ CFU/ml of L. monocytogenes suspended in BHI or minimal medium was placed on the stainless steel surface. After holding in saturated humidity for 3 h at the desired temperature the surface was washed and prepared for scanning electron microscopy (SEM). Using SEM, attachment of L. monocytogenes was determined by counting cells remaining on the surface. When L. monocytogenes cultivated in BHI were used, with the exception of the number of attached cells being lower at 42 °C than at 37 °C and 30 °C, the number of attached cells increased with increasing temperature (P < 0.05). When L. monocytogenes starved in minimal medium were used, the number of attached cells also increased with increasing attachment temperature (P < 0.05), but the number of attached cells at 42 °C was lower than that at the other temperatures. The attachment of L. monocytogenes to stainless steel surface was greater when cultivated in rich medium of BHI vs starved in the minimal medium.     

Marked intra-strain variation in response of Listeria monocytogenes dairy isolates to acid or salt stress and the effect of acid or salt adaptation on adherence to abiotic surfaces

During food processing, and particularly in cheese manufacturing processes, Listeria monocytogenes may be exposed routinely to environments of low pH or high salt concentration. It has been suggested that these environmental conditions may contribute to bacterial adherence to abiotic surfaces and increased resistance to disinfection. In this study strains isolated from the environment of artisanal cheese-making dairies were used to investigate the behaviour of L. monocytogenes in response to acid and salt stress and clear differences between strains was observed. In planktonic culture, strains varied in resistance to low pH or high NaCl concentration and in the occurrence of an adaptive response to moderate acid or NaCl. There was dislocation in responses to salt and acid. Strains resistant, or adaptive, to acid were not resistant or adaptive to NaCl. The reverse also was observed. Exposure to moderate acid did not promote adherence to polystyrene but survival, at low pH or high NaCl concentration, of cells adherent to stainless steel was increased, even for strains that had no adaptive response planktonically, but the detail of these observations varied between strains. In contrast to acid adaptation, with some strains salt adaptation enhanced adherence of L. monocytogenes to polystyrene but this was not true for all strains. For some strains salt- or acid adaptation may enhance the survival of sessile cells exposed to hypochlorite disinfection.     

Monolaurin and acetic acid inactivation of Listeria monocytogenes attached to stainless steel.

Individual and combined antimicrobial effects of monolaurin and acetic acid on Listeria monocytogenes planktonic cells or stainless-steel-adherent cells were determined in order to evaluate cell viability during a 25-min exposure period at 25 degrees C. A 10(7)-colony-forming units (CFU)/ml population of planktonic cells was completely inactivated by the synergistic combination of 1% acetic acid with 50 or 100 microg/ml of monolaurin within 25 or 20 min, respectively. Either compound alone caused partial but incomplete inactivation within the same time periods. A population of 10(5) CFU/cm2 of 1-day adherent cells on stainless steel was completely inactivated within 25 min, but with the highest concentrations of the combined chemicals, i.e., 1% acetic acid and 100 microg/ml of monolaurin. The combined chemical treatment again synergistically produced greater inhibition. A 10(6)-CFU/cm2 population of 7-day adherent cells was not completely inactivated within 25 min of exposure, although counts did decline. The results demonstrate increased resistance of attached L. monocytogenes to acetic acid and monolaurin and show that resistance increased with culture age. Combinations of organic acids and monolaurin might be considered as sanitizers of food contact surfaces, but activities of such combinations are likely to be less than other commonly used sanitizers.     

Effect of surface roughness and stainless steel finish on Listeria monocytogenes attachment and biofilm formation

The purpose of this study was to evaluate the effect of surface roughness (Ra) and finish of mechanically polished stainless steel (Ra = 0.26 +/- 0.05, 0.49 +/- 0.10, and 0.69 +/- 0.05 microm) and electropolished stainless steel (Ra = 0.16 +/- 0.06, 0.40 +/- 0.003, and 0.67 +/- 0.02 microm) on Listeria adhesion and biofilm formation. A four-strain cocktail of Listeria monocytogenes was used. Each strain (0.1%) was added to 200 ml of tryptic soy broth (TSB), and coupons were inserted to the mixture for 5 min. For biofilm formation, coupons with adhesive cells were incubated in 1:20 diluted TSB at 32 degrees C for 48 h. The experiment was performed by a randomized block design. Our results show that the level of Listeria present after 48 h of incubation (mean = 7 log CFU/cm2) was significantly higher than after 5 min (mean = 6.0 log CFU/cm2) (P < 0.01). No differences in initial adhesion were seen in mechanically finished (mean = 6.7 log CFU/cm2) when compared with electropolished stainless steel (mean = 6.7 log CFU/cm2) (P > 0.05). Listeria initial adhesion (values ranged from 5.9 to 6.1 log CFU/cm2) or biofilm formation (values ranged from 6.9 to 7.2 log CFU/cm2) was not significantly correlated with Ra values (P > 0.05). Image analysis with an atomic force microscope showed that bacteria did not colonize the complete surface after 48 h but were individual cells or grouped in microcolonies that ranged from 5 to 10 microm in diameter and one to three cell layers in thickness. Exopolymeric substances were observed to be associated with the colonies. According to our results, electropolishing stainless steel does not pose a significant advantage for food sanitation over mechanically finished stainless steel.     

Heat and acid tolerance of Listeria monocytogenes after exposure to single and multiple sublethal stresses

The majority of published studies on the adaptive heat or acid tolerance response of Listeria monocytogenes have been performed with a single strain exposed to a single adaptation treatment; however, in food ecosystems, microorganisms commonly exist as multi-species communities and encounter multiple stresses, which may result in "stress hardening". Therefore, the present study evaluated the adaptive responses to heat (52, 57 and 63 degrees C) or lactic acid (pH 3.5) of a 10-strain composite of L. monocytogenes meat and human isolates at stationary phase, following exposure to combinations of osmotic (10% NaCl), acidic (pH 5.0 with HCl) and thermal (T; 46 degrees C) stresses, sequentially or simultaneously within 1.5h, in tryptic soy broth with 0.6% yeast extract (TSBYE). All treatments induced adaptive responses on L. monocytogenes at 57 degrees C, while no such cross-protection was observed at 52 and 63 degrees C. Survivor curves at 57 degrees C appeared convex with profound shoulders determined by a Weibull model. The highest thermotolerance was observed after combined exposure to acid and heat shock (pH-T), followed by exposure to osmotic shock, and by the combination of osmotic with heat shock (NaCl-T). Regarding acid tolerance, prior exposure to low pH, pH-T, or a combination of NaCl, pH and T resulted in a marked increase of resistance to pH 3.5, showing concave inactivation curves with tails at higher levels of survivors (log(10)CFU ml(-1)) than the control cultures. The sequence of exposure to sublethal stresses did not affect the thermotolerance of L. monocytogenes, whereas simultaneous exposure to most multiple stresses (e.g., NaCl-pH-T, NaCl-T and NaCl-pH) resulted in higher survivors of L. monocytogenes at pH 3.5 than exposure to the same stresses sequentially. The results indicate that combinations and sequences of sublethal hurdles may affect L. monocytogenes acid and heat tolerance, especially in acidic environments with mild heating or in low moisture environments.     

Survival of Listeria monocytogenes attached to stainless steel surfaces in the presence or absence of Flavobacterium spp

Contaminated surfaces of food processing equipment are believed to be a significant source of Listeria monocytogenes to foods. However, very little is known about the survival of Listeria in processing environments. In a mixed bacterial biofilm of L. monocytogenes and Flavobacterium spp., the number of L. monocytogenes cells attaching to stainless steel increased significantly compared to when L. monocytogenes was in a pure culture. The L. monocytogenes cells in the mixed biofilms were also recoverable for significantly longer exposure periods. On colonized coupons held at 15 degrees C and 75% humidity, decimal reduction times were 1.2 and 18.7 days for L. monocytogenes in pure and mixed biofilms, respectively. With increasing exposure time, the proportion of cells that were sublethally injured (defined as an inability to grow on selective agar) increased from 8.1% of the recoverable cell population at day 0 to 91.4% after 40 days' exposure. At 4 and -20 degrees C, decimal reduction times for L. monocytogenes in pure culture were 2.8 and 1.4 days, respectively, and in mixed culture, 10.5 and 14.4 days, respectively. The enhanced colonization and survival of L. monocytogenes on "unclean" surfaces increase the persistence of this pathogen in food processing environments, while the increase in the percentage of sublethally injured cells in the population with time may decrease the ability of enrichment regimes to detect it.     

Role of sigma B factor in the alkaline tolerance response of Listeria monocytogenes 10403S and cross-protection against subsequent ethanol and osmotic stress

Many of the considerable abilities of Listeria monocytogenes to persist and grow in a wide range of adverse environmental conditions are thought to be at least partly under the control of the alternative sigma factor (sigmaB), encoded by the sigB gene. However, little is known about the role of this master regulon in the impressive ability of Listeria to persist and grow under conditions of alkaline pH. In this study, Northern blot analysis of parent Listeria mRNA revealed that alkali adaptation (pH 9.5 for 1 h) significantly increased the expression of sigB-derived mRNA. The study included a comparison of the relative survival of mid-exponential populations of adapted and nonadapted parent type (sigmaB expressing) and mutant (not sigmaB expressing, deltasigB) Listeria strains during subsequent alkaline (pH 12.0), osmotic (25% NaCl, wt/vol), or ethanol (16.5%) stress. Alkali-adapted parent strains were more resistant to pH 12.0 than were adapted deltasigB type strains, but both alkali-adapted parent and deltasigB strains were more resistant to pH 12.0 than were nonadapted strains. Alkali-adapted parent strains were more resistant to osmotic stress than were adapted deltasigB type strains. No significant differences in viability were observed between alkali-adapted parent and deltasigB strains after ethanol stress, suggesting that cross-protection against osmotic stress is mediated by sigmaB whereas cross-protection against ethanol is sigmaB independent. Overall, alkali-induced cross-protection against osmotic and ethanol challenges may have serious implications for food safety and human health because such stress conditions are routinely used as part of food preservation and surface cleaning processes.     

Attachment of Listeria monocytogenes to an austenitic stainless steel after welding and accelerated corrosion treatments

Austenitic stainless steels, widely used in food processing, undergo microstructural changes during welding, resulting in three distinctive zones: weld metal, heat-affected zone, and base metal. This research was conducted to determine the attachment of Listeria monocytogenes in these three zones before and after exposure to a corrosive environment. All experiments were done with tungsten inert gas welding of type 304 stainless steel. The four welding treatments were large or small beads with high or low heat. After welding, all surfaces were polished to an equivalent surface finish. A 10-microl droplet of an L. monocytogenes suspension was placed on the test surfaces. After 3 h at 23 degrees C, the surfaces were washed and prepared for scanning electron microscopy, which was used to determine attachment of L. monocytogenes by counting cells remaining on each test surface. In general, bacteria were randomly distributed on each surface type. However, differences in surface area of inoculum due to differences in interfacial energy (as manifested by the contact angle) were apparent and required normalization of bacterial count data. There were no differences (P > 0.05) in numbers of bacteria on the three surface zones. However, after exposure to the corrosive medium, numbers of bacteria on the three zones were higher (P < 0.05) than those on the corresponding zones of noncorroded surfaces. For the corroded surfaces, bacterial counts on the base metal were lower (P < 0.05) than those on heat-affected and weld zones.     

Effects of the growth procedure on the surface hydrophobicity of Listeria monocytogenes cells and their adhesion to stainless steel.

The aim of this study was to examine the physicochemical surface properties and the ability to adhere to stainless steel of three strains of Listeria monocytogenes after different cultivation procedures. To this end, bacteria were cultivated at 37 degrees C after storage at two frequently used temperatures (4 degrees C or -80 degrees C) and were then transferred into the liquid medium (trypticase soy broth supplemented with 6 g liter(-1) of yeast extract, pH 7.3) between one and four times. In addition, the influence of supplementing the growth medium with lactic acid was explored, this organic acid being representative of both the dairy and cured meat industries. The hydrophobic/hydrophilic and electron-acceptor/electron-donor characteristics of the strains were evaluated by the microbial adhesion to solvents method. Using this technique, we recorded an increase in the hydrophobic properties of one strain stored at 4 degrees C, with an increasing number of transfers in the media (P < 0.05). Another plant-isolated strain appeared more hydrophobic and stuck better to stainless steel when cells were stored at 4 degrees C rather than at -80 degrees C. Preculturing L. monocytogenes in a lactic acid-supplemented medium increased the affinity of microbial cells to solvents and the bacterial attachment to stainless steel (P < 0.05).     

Survival of osmotic and acid stress by Listeria monocytogenes strains of clinical or meat origin

The ability of 30 Listeria monocytogenes strains, 15 of meat origin and 15 of clinical origin, to use carnitine as an osmoprotectant and to resist acid stress was determined. All strains examined were able to use carnitine as an osmoprotectant, indicating the importance of this characteristic to the survival of L. monocytogenes in natural environments. Clinical and meat strains, however, differed with respect to this characteristic. Specifically, 73% of meat strains reached a lower maximum cell density in the presence of carnitine with osmotic stress than in its absence with no stress. Only 33% of clinical strains displayed the same feature whereas the remaining clinical strains reached a higher maximum cell density in the presence of carnitine with osmotic stress than in its absence with no stress. The physiological reasons and advantage of this difference are unclear. When exposed to conditions of severe acid stress (pH 2.5) for 2 h, only two L. monocytogenes strains (L66 and L78), both of meat origin, displayed significant reductions (P < 0.05) in number (3.51 and 2.79 log cfu, respectively). Acid-sensitive strains were not found among the clinical isolates examined, highlighting the importance of acid stress resistance in the infection process.     

Desiccation of adhering and biofilm Listeria monocytogenes on stainless steel: Survival and transfer to salmon products

The foodborne bacterial pathogen, Listeria monocytogenes, commonly contaminates foods during processing, where the microorganisms are potentially subjected to low relative humidity (RH) conditions for extended periods of time. The objective of this study was to examine survival during desiccation (43% RH and 15 °C) of biofilm L. monocytogenes N53-1 cells on stainless steel coupons and to assess subsequent transfer to salmon products. Formation of static biofilm (2 days at 100% RH and 15 °C) prior to desiccation for 23 days significantly (P < 0.05) improved survival of cells desiccated in initial low salt concentrations (0.5%) compared to the survival for non-biofilm cells also desiccated in low salt, indicating the protective effect of the biofilm matrix. Osmoadaptation of cells in 5% NaCl before formation of the static biofilm significantly (P < 0.05) increased long-term desiccation survival (49 days) irrespectively of the initial salt levels (0.5% and 5% NaCl). The efficiency of transfer (EOT) of desiccated biofilm cells was significantly (P < 0.05) lower than EOTs for desiccated non-biofilm bacteria, however, as biofilm formation enhanced desiccation survival more bacteria were still transferred to smoked and fresh salmon. In conclusion, the current work shows the protective effect of biofilm formation, salt and osmoadaptation on the desiccation survival of L. monocytogenes, which in turn increases the potential for cross-contamination during food processing.     

The resistance to detachment of dairy strains of Listeria monocytogenes from stainless steel by shear stress is related to the fluid dynamic characteristics of the location of isolation

Strains of Listeria monocytogenes isolated from artisanal Portuguese cheese-making dairies were divided into two categories on the basis of the locations from which they were isolated: strains from dynamic locations were those that were habitually exposed to flowing liquids during the process of cheese-making, whereas those from static locations were rarely, if ever, exposed to the shear stresses generated by liquid flows. The strength of attachment to stainless steel discs of all of these strains was obtained using a radial flow chamber. Initial attachment strengths to stainless steel (after a 0.5 h contact time) of L. monocytogenes strains were greater for the 5 isolates from surfaces exposed to flow (dynamic isolates) than for most (3 out of 4) of those that were not (static isolates). After a 24 h contact time, attachment strength of all isolates reached similar levels. These results suggest that strains having high initial attachment strength are more likely to persist on surfaces exposed to flow than strains having low initial attachment strength. The numerical values of shear forces obtained could prove useful in the rational design of cleaning and decontamination procedures in food processing facilities.     

Bactericidal activity of strong acidic hypochlorous water against Escherichia coli O157:H7 and Listeria monocytogenes in biofilms attached to stainless steel

Food Science and Biotechnology - This study aims to investigate the bactericidal activity of strong acidic hypochlorous water (SAHW) against Escherichia coli O157:H7 and L. monocytogenes in...     

Evaluation of the transfer of Listeria monocytogenes from stainless steel and high-density polyethylene to Bologna and American cheese

The objective of this study was to determine the factors involved in the transfer of Listeria monocytogenes from surfaces to foods. We evaluated the influence of surface type (stainless steel and high-density polyethylene), inoculation method (biofilm growth and attached cells), hydration level (visibly dry and wet), and food type (bologna and American cheese). Each experiment included all 16 combinations and was repeated 11 times. A four-strain cocktail of L. monocytogenes was used to inoculate stainless steel and high-density polyethylene either as growing biofilms or attached cells. Slides were placed on a universal testing machine and brought into contact with food at a constant pressure (45 kPa) and time (30 s). Food slices were blended, the number of transferred cells was determined by plating, and the efficiency of transfer (EOT) was calculated. The results strongly suggest that stainless steel surfaces transferred more L. monocytogenes to foods than did polyethylene (P = 0.05). Independent of the surface, biofilms tended to transfer more L. monocytogenes to foods (EOT = 0.57) than did attached cells (EOT = 0.16). Among foods, L. monocytogenes was transferred to bologna more easily than to cheese (P < 0.05). The impact of hydration on transfer was significantly higher for dried biofilms growing on stainless steel (P < 0.05). No significant differences for hydration were seen under other conditions (P > 0.05). We hypothesize that drying weakens cell-to-cell interactions in biofilms and cell-to-surface interactions of biofilms and thus allows increased transfer of cells to food products.     

Evaluation of growth/no growth interface of Listeria monocytogenes growing on stainless steel surfaces, detached from biofilms or in suspension, in response to pH and NaCl

The present study aimed to describe the growth/no growth interface of Listeria monocytogenes at three potential states of growth in industrial environments, namely attached, (Att), detached (Det) from a biofilm, or in a planktonic state (suspended; Plan). A 3-strain composite of L. monocytogenes cells was left to colonize stainless steel (SS) surfaces in tryptic soy broth supplemented with 0.6% yeast extract (TSBYE) at 20 °C for 72 h and then transferred to TSBYE at 30 different pH and NaCl concentrations, which were renewed every two days during incubation at 10 °C. Survival of attached population was observed at optimal conditions (pH 7.2, a(w) 0.996), whereas at 4.5-8.0% salt and/or pH<6.0, reduction of attached population on SS surfaces was observed. PFGE patterns showed that 91% of the cells colonizing the SS coupons after 30 days, at any pH and a(w) conditions, belonged to a single strain. Furthermore, the change in the probability of a single cell to initiate growth (P(in)) over time, as well as the number of cells needed (CN) for growth initiation of planktonically growing Plan and Det L. monocytogenes cells were evaluated based on MPN Tables. An ordinary logistic regression model was also used to describe the growth/no growth interface of varying inoculation levels (from <10 to 10(4)CFU/ml) of Plan and Det cells in response to pH and a(w). Although both cell types demonstrated similar growth limits at populations of 10(2)-10(4)CFU/ml, higher numbers of Det than Plan cells were needed (CN) in order to initiate growth at low a(w) and pH. Individual Plan cells reached higher maximum levels of probability of growth initiation (P(max)) and had shorter times to reach P(max)/2 (t(au)), compared to their Det counterparts. Data on growth potential of cells in suspension, attached or detached status, may assist in ranking the risk from different sources of contamination. In addition, they may establish the link between the behavior of L. monocytogenes in foods and its origin from the processing plant. The latter link is important component of biotraceability.     

Survival of Listeria monocytogenes strain H7762 and resistance to simulated gastric fluid following exposure to frankfurter exudate

Listeria monocytogenes strain H7762, a frankfurter isolate, was tested to determine whether it was able to survive at 4 degrees C in frankfurter pack fluid (exudate) and to determine whether food exposure affects its acid sensitivity. Cultures were sampled and tested for acid sensitivity by challenge with simulated gastric fluid (SGF). SGF challenges performed immediately after inoculation revealed that between 20 and 26% of the cells survived the full 30 min of SGF challenge regardless of whether the cells were inoculated into brain heart infusion broth (BHI) or exudate. After 2 days of incubation, cells exposed to both exudate and BHI had significantly decreased SGF resistance; however, the cells exposed to exudate were significantly more SGF resistant than cells exposed to BHI (after 15 min of SGF treatment, 33% of the exudate-exposed cells survived and 12% of the BHI-exposed cells survived). L. monocytogenes exposed to exudate had greater SGF resistance at all challenge times compared with BHI-exposed cells from day 2 through day 4. From days 8 to 15, exudate-exposed cells continued to have greater SGF resistance than BHI-exposed cells up to 10 min of SGF challenge but were as sensitive as the BHI-exposed cells at 20 to 30 min of challenge. By day 25, cells exposed to exudate were significantly more sensitive to SGF challenge than BHI-exposed cells. The survivor data generated from SGF challenges were modeled by a nonlinear regression analysis to calculate the underlying distribution of SGF resistance found in the challenged populations. These analyses indicated that L. monocytogenes exposed to exudate at 4 degrees C had a broader distribution of resistance to SGF compared with cells exposed to BHI at 4 degrees C. In addition, the mean time of death during SGF treatment was greater after exposure to exudate, indicating that cells exposed to exudate were more resistant to killing by SGF These data suggest that exposure to frankfurter exudate might render L. monocytogenes more able to survive the stomach environment during the initial stages of infection.     

Effect of biofilm dryness on the transfer of Listeria monocytogenes biofilms grown on stainless steel to bologna and hard salami

Listeria monocytogenes continues to be a major cause of class I food recalls in the United States. Very little is known about its transfer and cross-contamination in processing scenarios. The objective of this study was to evaluate the effect of hydration level on L. monocytogenes biofilms grown on stainless steel and its effect on the biofilm transfer to foods. Biofilms were grown on stainless steel in diluted tryptic soy broth 1:20 for 48 h at 32 degrees C. After this, biofilms were equilibrated over saturated salt solutions at 20 degrees C for 24 h (94, 75, 58, and 33% relative humidity; % RH) prior to transferring. Transfer experiments were conducted from inoculated stainless steel to bologna and hard salami at a constant pressure (45 kPa) and time (30 s) with a universal testing machine. The experiment was designed with a factorial design 4 x 2 (biofilms equilibrated at 4% RH and two foods) and duplicated every day, and the whole experiment was repeated nine times. The results were analyzed with an analysis of variance by SAS Statistical Analysis Software. Our results showed that more bacteria were transferred to bologna (mean efficiency of transfer [EOT] = 3.0) than to hard salami (mean EOT = 0.35, P < 0.01). As biofilms became drier, the transfer of Listeria from stainless steel to both foods increased (P < 0.05). The EOT increased from 2 to 3.8 and from 0.2 to 0.51 upon transfer when drying the biofilm for bologna and hard salami, respectively. This study may be an indication that as biofilms were dried, the cell-cell and cell-surface interactions became weaker, and bacterial transfer increased. This phenomenon was enhanced in foods containing higher water activity levels. We hypothesize that this increased in transfer was due to the presence of capillary forces in the food.     

Role of uvrA in the growth and survival of Listeria monocytogenes under UV radiation and acid and bile stress

Listeria monocytogenes encounters numerous stresses both in the food environment and during infection of the host. The ability to survive and tolerate bile and low pH conditions, which are two major stresses, is of particular importance for survival within the host. The uvrA gene in other bacteria is involved in the repair of acid-induced DNA damage and in adaptation to low pH. Thus, a uvrA in-frame deletion mutant was constructed to identify the role of uvrA in the growth and survival of L. monocytogenes under various environmental conditions. The uvrA mutant was highly sensitive to UV radiation. Growth under normal laboratory conditions was impaired during the exponential phase, and the time to reach the exponential phase of growth, TV(max), was significantly delayed (P < 0.05). Growth of the uvrA mutant in acidic medium (pH 5) was slightly impaired, and the TV(max) was significantly delayed (P < 0.05). Growth and the TV(max) of the mutant in the presence of 0.3% bile salts also were significantly impaired (P < 0.05). These results suggest that uvrA is needed for optimal growth and survival of L. monocytogenes under various stressful environmental conditions.     

Influence of flow direction and flow rate on the initial adhesion of seven Listeria monocytogenes strains to fine polished stainless steel

The effects of flow direction and shear stress on the adhesion of different strains of Listeria monocytogenes to fine polished stainless steel under liquid flow conditions were investigated. Furthermore, the relationship between cell surface properties and cell size and the initial adhesion rate (IAR) was studied. A method, including fluorescence microscopy and a flow perfusion system, was developed and used to examine the real-time initial cell adhesion of different L. monocytogenes species in situ to opaque surfaces under flow conditions. The results demonstrated that shear stress was the determining factor for the initial adhesion of L. monocytogenes under flow conditions. The flow direction in relation to the orientation of surface features (the scratches) could be disregarded. IARs were dependent on the shear stress and strain type. The strain EGDe, which had the lowest IAR, had the largest cell size, was the least hydrophobic and possessed the most electron-donating cell surface. Except for the L. monocytogenes strain EGDe, no clear correlations were found between the IAR and cell surface properties, or cell size. In conclusion, many factors may be involved in determining the initial adhesion of L. monocytogenes to stainless steel under flow conditions. Two of the main factors are flow rate/shear stress and strain specificity.     

Mixed species biofilms of Listeria monocytogenes and Lactobacillus plantarum show enhanced resistance to benzalkonium chloride and peracetic acid

We investigated the formation of single and mixed species biofilms of Listeria monocytogenes strains EGD-e and LR-991, with Lactobacillus plantarum WCFS1 as secondary species, and their resistance to the disinfectants benzalkonium chloride and peracetic acid. Modulation of growth, biofilm formation, and biofilm composition was achieved by addition of manganese sulfate and/or glucose to the BHI medium. Composition analyses of the mixed species biofilms using plate counts and fluorescence microscopy with dual fluorophores showed that mixed species biofilms were formed in BHI (total count, 8-9 log₁₀ cfu/well) and that they contained 1-2 log₁₀ cfu/well more L. monocytogenes than L. plantarum cells. Addition of manganese sulfate resulted in equal numbers of both species (total count, 8 log₁₀ cfu/well) in the mixed species biofilm, while manganese sulfate in combination with glucose, resulted in 1-2 log₁₀ more L. plantarum than L. monocytogenes cells (total count, 9 log₁₀ cfu/well). Corresponding single species biofilms of L. monocytogenes and L. plantarum contained up to 9 log₁₀ cfu/well. Subsequent disinfection treatments showed mixed species biofilms to be more resistant to treatments with the selected disinfectants. In BHI with additional manganese sulfate, both L. monocytogenes strains and L. plantarum grown in the mixed species biofilm showed less than 2 log₁₀ cfu/well inactivation after exposure for 15 min to 100 μg/ml benzalkonium chloride, while single species biofilms of both L. monocytogenes strains showed 4.5 log₁₀ cfu/well inactivation and single species biofilms of L. plantarum showed 3.3 log₁₀ cfu/well inactivation. Our results indicate that L. monocytogenes and L. plantarum mixed species biofilms can be more resistant to disinfection treatments than single species biofilms.